Rat liver microsomal fixed function oxidase catalyses the hydroxylation of the cyclohexyl moiety of 1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea (CCNU to give at least five metabolites. When exposed to alkaline pH at 100 degrees, CCNU and its metabolites quantitatively release their cyclohexyl moiety as cyclohexylamine and aminocyclohexanol, respectively. The N-(2,4-dinitrophenyl) derivatives of cyclohexylamine and aminocyclohexanols were separated by high pressure liquid chromatography. The in vitro metabolites have been identified as trans-2-hydroxy CCNU, cis-3-hydroxy CCNU, trans-3-hydroxy CCNU, cis-4-hydroxy CCNU and trans-4-hydroxy CCNU. Ring hydroxylation axial to the 1-(2-chloroethyl)-1-nitrosourea group (cis-2-trans-3-,cis-4-) is favored over equatorial attack (trans-2-, cis-3-, trans-4). This project is progressing toward the quantitative preparation of stable derivatives of the nitrosoureas and their hydroxylated metabolites in order to facilitate future pharmacological studies in experimental animals and man.